Mechanical constraints to unbound expansion of B. subtilis on semi-solid surfaces

ABSTRACT The effects of surface mechanical constraints that may promote or prevent bacterial expansion on semi-solid surfaces are largely unknown. In this work, we have manufactured agar surfaces with different viscoelasticity, topography, and roughness. To capture the essential biophysics of the bacterial expansion we have developed a continuum model that faithfully reproduces the main patterns of the short-range and long-range expansion with two critical parameters: local interfacial forces and colony viscosity. Cohesive energy of the bacterial colony that determines the extent of exploration was dependent on agar surface viscoelasticity. On soft surfaces, bacteria produce low viscoelastic colonies that allow guided population of bacteria to traverse distances that are six orders of magnitude larger than the size of the individual bacterium. Bacteria growing on stiff surfaces produce colonies with significantly increased viscoelasticity that prevent bacterial exploration of new territory and allow formation of a very steep cliff at the edge of the colony. Upon flooding of the rough surfaces, we have induced aquaplaning and spreading of bacteria. A layer of water between the bacterium and surface results in a loss of traction allowing bacteria to spread across the otherwise inhibitory rough surface. The results shed new light on the bacterial ability to rapidly colonize new territories. IMPORTANCE How bacterial cells colonize new territory is a problem of fundamental microbiological and biophysical interest and is key to the emergence of several phenomena of biological, ecological, and medical relevance. Here, we demonstrate how bacteria stuck in a colony of finite size can resume exploration of new territory by aquaplaning and how they fine tune biofilm viscoelasticity to surface material properties that allows them differential mobility. We show how changing local interfacial forces and colony viscosity results in a plethora of bacterial morphologies on surfaces with different physical and mechanical properties.

The authors present a characterization of the physical and abiotic parameters (roughness, roughness, extracellular matrix components and viscoelasticity of the biofilm) that affect the surface-associated mobility of the model bacterium Bacillus subtilis.This behavior is monitored as the concentrations of agar in the culture medium and the supply of nutrients change.The work is interesting from the basic point of view to begin to understand the way in which factors of the extracellular environment affect the ability of a bacterium to move.The work is well written, it is understandable both for a non-expert in Microbiology and for a nonexpert in Physics.However, there are some "minor" points to clarify.
1-Is the B. subtilis strain used by the authors (PS-216 strain) derived from the NCIB3610 strain?The reference (reference #1) that the authors give for this strain corresponds to a review on different forms of bacterial mobility and I could not find any mention of the B.subtilis PS-216 strain.Could the authors indicate the origin of this strain or give an appropriate reference?2-If the PS-216 strain derives from the NCIB3610 strain or another non-domesticated strain, it would be a prototrophic strain, so why to work with the MSgg medium with the Trp and Phe amino acid aggregates that are necessary for the growth of the domesticated strain JH642 but not for strain NCIB3610 or strains derived from it.In line 98 it should not be indicated that it is a minimal medium (if the mentioned amino acids are not essential for the growth of the strain used in this work).
3-The use of the term "exploratory" is not clear to me since it could give the idea that it is a "back and forth" movement of the bacterium from the colony ("out") and back ( "back") to it after exploring the territory.The closest thing that comes to mind now would be the adventurous movement (A motility)of Mixococcus xantus as opposed to its social movement (S Motility).Perhaps the use of the word "exploratory" could be replaced with something else like "social" or "multicellular" movement.

4-In fig S4
, it is shown that the production of surfactin and EPS is maximum when the colony (solid biofilm) develops on MSgg plates with agar at high concentrations (6%).Since the motility mechanism described by the authors (although they do not state it explicitly) would be dependent on the flagellum (swarming motility) at this high concentration of agar (4 -6%), the overproduction of EPS and surfactin could indicate one or two alternatives: biofilm formation is favored (which requires surfactin and EPS) and/or the social mechanism of sliding displacement on surfaces would be activated at later times, to which the authors do not refer.Another alternative, not exclusive of the above, could be that the overproduction of EPS and surfactin would allow several layers of cells to form, one on top of the other, as opposed to the monolayer of mobile cells on a surface with a low concentration of agar ( 0.75%) or other types of obstacles.A mention should be made to these alternatives between biofilm formation and social sliding.

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Point by point reply to reviewers' comments
Reviewer #1 (Comments for the Author): In the manuscript the authors set out to elucidate bacteria's ability to explore and colonize its environment.They investigated the effect of surface mechanical properties (as defined by the differing viscoelastic properties of the agar surface that translated into surface roughness and topology) on B. subtilis's ability to spread over the agar surface.They also investigated the contribution of biofilm and surfactant production on the ability of B. subtilis to explore and colonize its immediate environment.Finally, they explored whether proposed genetic deletions could be overcome by external addition of surfactants or water through the phenomenon of aquaplaning.Overall, by examining the viscoelastic properties of the bacterial biofilm and the corresponding relationship to surface stiffness, the manuscript sheds new insights into how the long-range exploratory bacterial growth occurs and that it is intimately dependent on the physical properties of its immediate environment.The manuscript is well written and logically structured.The experiments are technically sound and the results support the presented conclusions of the work.The research would be of interest to the scientific community, is novel and is in Microbiology Spectrum's scope.
We would like to thank the reviewer for the encouraging words.

Reviewer #2 (Comments for the Author):
The authors present a characterization of the physical and abiotic parameters (roughness, roughness, extracellular matrix components and viscoelasticity of the biofilm) that affect the surface-associated mobility of the model bacterium Bacillus subtilis.This behavior is monitored as the concentrations of agar in the culture medium and the supply of nutrients change.The work is interesting from the basic point of view to begin to understand the way in which factors of the extracellular environment affect the ability of a bacterium to move.The work is well written, it is understandable both for a non-expert in Microbiology and for a non-expert in Physics.However, there are some "minor" points to clarify.
We appreciate that the reviewer finds our work "interesting" and "well-written".We would also like to thank the reviewer for providing constructive feedbacks, which helped us improve the manuscript.

1-Is the B. subtilis strain used by the authors (PS-216 strain) derived from the NCIB3610 strain? The reference (reference #1) that the authors give for this strain corresponds to a review on different forms of bacterial mobility and I could not find any mention of the B.subtilis PS-216 strain. Could the authors indicate the origin of this strain or give an appropriate reference?
B. subtilis strain PS-216 is not a derivate of NCIB3610 strain.It is a natural isolate of B. subtilis, obtained from the sandy soil samples on the bank of the River Sava, Slovenia (grid reference 46°06′N, 14°28′E) in January 2006.The bacterial strain was first described in the work of Stefanic P, Mandic-Mulec I. 2009 entitled "Social interactions and distribution of Bacillus subtilis pherotypes at microscale" in J Bacteriol 191:1756-1764 (Supplementary Ref. 1 of the original manuscript) and has been used ever since in our laboratory.

Revisions:
We have cited the paper mentioned above in the revised manuscript and clearly stated the origin of the strain in the main text.
Lines 367-368: B. subtilis strain PS-216 is a natural isolate of B. subtilis, obtained from the sandy soil samples on the bank of the River Sava (49).
2-If the PS-216 strain derives from the NCIB3610 strain or another non-domesticated strain, it would be a prototrophic strain, so why to work with the MSgg medium with the Trp and Phe amino acid aggregates that are necessary for the growth of the domesticated strain JH642 but not for strain NCIB3610 or strains derived from it.In line 98 it should not be indicated that it is a minimal medium (if the mentioned amino acids are not essential for the growth of the strain used in this work).

Revision:
In line 109 of the revised manuscript, we have removed "minimal" as suggested by the reviewer.
3-The use of the term "exploratory" is not clear to me since it could give the idea that it is a "back and forth" movement of the bacterium from the colony ("out") and back ( "back") to it after exploring the territory.The closest thing that comes to mind now would be the adventurous movement (A motility)of Mixococcus xantus as opposed to its social movement (S Motility).Perhaps the use of the word "exploratory" could be replaced with something else like "social" or "multicellular" movement.
We thank the reviewer for this suggestion.We agree that exploratory growth might be ambiguous in the sense explained by the reviewer.

Revisions:
To make this clear we have replaced exploratory growth with expansion throughout the text.The expansion describing spreading of bacteria on solid surfaces has been used in the literature before (e.g., doi: 10.1039/d0sm01348j).

4-In fig S4
, it is shown that the production of surfactin and EPS is maximum when the colony (solid biofilm) develops on MSgg plates with agar at high concentrations (6%).Since the motility mechanism described by the authors (although they do not state it explicitly) would be dependent on the flagellum (swarming motility) at this high concentration of agar (4 -6%), the overproduction of EPS and surfactin could indicate one or two alternatives: biofilm formation is favored (which requires surfactin and EPS) and/or the social mechanism of sliding displacement on surfaces would be activated at later times, to which the authors do not refer.Another alternative, not exclusive of the above, could be that the overproduction of EPS and surfactin would allow several layers of cells to form, one on top of the other, as opposed to the monolayer of mobile cells on a surface with a low concentration of agar ( 0.75%) or other types of obstacles.A mention should be made to these alternatives between biofilm formation and social sliding.
We thank the reviewer for bringing up the different possibilities.
Our results show that bacteria try to overcome mechanical constraints on higher agar concentrations by increased production of surfactin.Whereas surfactin should facilitate colony expansion, the concomitant increase in EpsA-O polysaccharide promotes biofilm formation and inhibits colony expansion.The results on higher agar surfaces suggest that biofilm formation prevails, as the colonies are unable to expand laterally under such conditions (Fig. 1) and can only grow vertically to form structures that are several cell layers thick (Fig. 4c), as opposed to the monolayer structures formed by swarming cells on a surface with a low concentration of agar (Fig. 4a).This may also provide a mechanical explanation why researchers have been studying swarming motility at relatively low agar concentrations (between 0.3 % to 1.0 %; see e.g., doi: 10.1038/nrmicro2405).
We did not think the restricted colony expansion at high agar concentrations is due to delayed activation of social sliding because we did not observe expansion of colonies on stiff agar even after a prolonged incubation (> 3 days).

Revisions:
We have modified the text to bring forward this point more clearly.
Lines 171-175: The increased expression of EpsA-O polysaccharide at higher agar concentrations correlates with biofilm formation several layers thick, as opposed to the monolayer structures of mobile cells on a surface with a low concentration of agar.Even after a prolonged incubation bacterial lateral expansion was halted.

Revisions:
We have corrected this in the revised manuscript.Two final comments need to be addressed in a minor text revision: In Material and Methods (lines 374 to 377), the names of the used restriction enzymes must be in italics.
-Line 371, use "lincomycin 12.5 ug/ml" instead "lincomycin 12,5 ug/ml" Thank you for submitting your manuscript to Microbiology Spectrum.When submitting the revised version of your paper, please provide (1) point-by-point responses to the issues raised by the reviewers as file type "Response to Reviewers," not in your cover letter, and (2) a PDF file that indicates the changes from the original submission (by highlighting or underlining the changes) as file type "Marked Up Manuscript -For Review Only".Please use this link to submit your revised manuscript -we strongly recommend that you submit your paper within the next 60 days or reach out to me.Detailed instructions on submitting your revised paper are below.

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Sincerely, Ilana Kolodkin-Gal
Editor, Microbiology Spectrum Journals Department American Society for Microbiology 1752 N St., NW Washington, DC 20036 E-mail: spectrum@asmusa.orgReviewer comments: Reviewer #2 (Comments for the Author): The authors have answered all concerns satisfactorily.The manuscript is now suitable for publication in Microbiology Spectrum.Minor points, -In Material and Methods (lines 374 to 377), the names of the used restriction enzymes must be in italics.

Preparing Revision Guidelines
To submit your modified manuscript, log onto the eJP submission site at https://spectrum.msubmit.net/cgi-bin/main.plex.Go to Author Tasks and click the appropriate manuscript title to begin the revision process.The information that you entered when you first submitted the paper will be displayed.Please update the information as necessary.Here are a few examples of required updates that authors must address: • Point-by-point responses to the issues raised by the reviewers in a file named "Response to Reviewers," NOT IN YOUR COVER LETTER.
• Upload a compare copy of the manuscript (without figures) as a "Marked-Up Manuscript" file.
• Each figure must be uploaded as a separate file, and any multipanel figures must be assembled into one file.For complete guidelines on revision requirements, please see the journal Submission and Review Process requirements at https://journals.asm.org/journal/Spectrum/submission-review-process.Submissions of a paper that does not conform to Microbiology Spectrum guidelines will delay acceptance of your manuscript." Please return the manuscript within 60 days; if you cannot complete the modification within this time period, please contact me.If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify me of your decision immediately so that the manuscript may be formally withdrawn from consideration by Microbiology Spectrum.
If your manuscript is accepted for publication, you will be contacted separately about payment when the proofs are issued; please follow the instructions in that e-mail.Arrangements for payment must be made before your article is published.For a complete list of Publication Fees, including supplemental material costs, please visit our website.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees.Need to upgrade your membership level?Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Microbiology Spectrum.

Point by point reply to reviewers' comments
Reviewer #1 (Comments for the Author): In the manuscript the authors set out to elucidate bacteria's ability to explore and colonize its environment.They investigated the effect of surface mechanical properties (as defined by the differing viscoelastic properties of the agar surface that translated into surface roughness and topology) on B. subtilis's ability to spread over the agar surface.They also investigated the contribution of biofilm and surfactant production on the ability of B. subtilis to explore and colonize its immediate environment.Finally, they explored whether proposed genetic deletions could be overcome by external addition of surfactants or water through the phenomenon of aquaplaning.Overall, by examining the viscoelastic properties of the bacterial biofilm and the corresponding relationship to surface stiffness, the manuscript sheds new insights into how the long-range exploratory bacterial growth occurs and that it is intimately dependent on the physical properties of its immediate environment.The manuscript is well written and logically structured.The experiments are technically sound and the results support the presented conclusions of the work.The research would be of interest to the scientific community, is novel and is in Microbiology Spectrum's scope.
We would like to thank the reviewer for the encouraging words.

Reviewer #2 (Comments for the Author):
The authors present a characterization of the physical and abiotic parameters (roughness, roughness, extracellular matrix components and viscoelasticity of the biofilm) that affect the surface-associated mobility of the model bacterium Bacillus subtilis.This behavior is monitored as the concentrations of agar in the culture medium and the supply of nutrients change.The work is interesting from the basic point of view to begin to understand the way in which factors of the extracellular environment affect the ability of a bacterium to move.The work is well written, it is understandable both for a non-expert in Microbiology and for a non-expert in Physics.However, there are some "minor" points to clarify.
We appreciate that the reviewer finds our work "interesting" and "well-written".We would also like to thank the reviewer for providing constructive feedbacks, which helped us improve the manuscript.
1-Is the B. subtilis strain used by the authors (PS-216 strain) derived from the NCIB3610 strain?The reference (reference #1) that the authors give for this strain corresponds to a review on different forms of bacterial mobility and I could not find any mention of the B.subtilis PS-216 strain.Could the authors indicate the origin of this strain or give an appropriate reference?B. subtilis strain PS-216 is not a derivate of NCIB3610 strain.It is a natural isolate of B. subtilis, obtained from the sandy soil samples on the bank of the River Sava, Slovenia (grid reference 46°06′N, 14°28′E) in January 2006.The bacterial strain was first described in the work of Stefanic P, Mandic-Mulec I. 2009 entitled "Social interactions and distribution of Bacillus subtilis pherotypes at microscale" in J Bacteriol 191:1756-1764 (Supplementary Ref. 1 of the original manuscript) and has been used ever since in our laboratory.

Revisions:
We have cited the paper mentioned above in the revised manuscript and clearly stated the origin of the strain in the main text.
Lines 367-368: B. subtilis strain PS-216 is a natural isolate of B. subtilis, obtained from the sandy soil samples on the bank of the River Sava (49).
2-If the PS-216 strain derives from the NCIB3610 strain or another non-domesticated strain, it would be a prototrophic strain, so why to work with the MSgg medium with the Trp and Phe amino acid aggregates that are necessary for the growth of the domesticated strain JH642 but not for strain NCIB3610 or strains derived from it.In line 98 it should not be indicated that it is a minimal medium (if the mentioned amino acids are not essential for the growth of the strain used in this work).

Revision:
In line 109 of the revised manuscript, we have removed "minimal" as suggested by the reviewer.
3-The use of the term "exploratory" is not clear to me since it could give the idea that it is a "back and forth" movement of the bacterium from the colony ("out") and back ( "back") to it after exploring the territory.The closest thing that comes to mind now would be the adventurous movement (A motility)of Mixococcus xantus as opposed to its social movement (S Motility).Perhaps the use of the word "exploratory" could be replaced with something else like "social" or "multicellular" movement.
We thank the reviewer for this suggestion.We agree that exploratory growth might be ambiguous in the sense explained by the reviewer.

Revisions:
To make this clear we have replaced exploratory growth with expansion throughout the text.The expansion describing spreading of bacteria on solid surfaces has been used in the literature before (e.g., doi: 10.1039/d0sm01348j).

4-In fig S4
, it is shown that the production of surfactin and EPS is maximum when the colony (solid biofilm) develops on MSgg plates with agar at high concentrations (6%).Since the motility mechanism described by the authors (although they do not state it explicitly) would be dependent on the flagellum (swarming motility) at this high concentration of agar (4 -6%), the overproduction of EPS and surfactin could indicate one or two alternatives: biofilm formation is favored (which requires surfactin and EPS) and/or the social mechanism of sliding displacement on surfaces would be activated at later times, to which the authors do not refer.Another alternative, not exclusive of the above, could be that the overproduction of EPS and surfactin would allow several layers of cells to form, one on top of the other, as opposed to the monolayer of mobile cells on a surface with a low concentration of agar ( 0.75%) or other types of obstacles.A mention should be made to these alternatives between biofilm formation and social sliding.
We thank the reviewer for bringing up the different possibilities.
Our results show that bacteria try to overcome mechanical constraints on higher agar concentrations by increased production of surfactin.Whereas surfactin should facilitate colony expansion, the concomitant increase in EpsA-O polysaccharide promotes biofilm formation and inhibits colony expansion.The results on higher agar surfaces suggest that biofilm formation prevails, as the colonies are unable to expand laterally under such conditions (Fig. 1) and can only grow vertically to form structures that are several cell layers thick (Fig. 4c), as opposed to the monolayer structures formed by swarming cells on a surface with a low concentration of agar (Fig. 4a).This may also provide a mechanical explanation why researchers have been studying swarming motility at relatively low agar concentrations (between 0.3 % to 1.0 %; see e.g., doi: 10.1038/nrmicro2405).
We did not think the restricted colony expansion at high agar concentrations is due to delayed activation of social sliding because we did not observe expansion of colonies on stiff agar even after a prolonged incubation (> 3 days).

Revisions:
We have modified the text to bring forward this point more clearly.
Lines 171-175: The increased expression of EpsA-O polysaccharide at higher agar concentrations correlates with biofilm formation several layers thick, as opposed to the monolayer structures of mobile cells on a surface with a low concentration of agar.Even after a prolonged incubation bacterial lateral expansion was halted.

Revisions:
We have corrected this in the revised manuscript.Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication.You will be notified when your proofs are ready to be viewed.
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ASM policy requires that data be available to the public upon online posting of the article, so please verify all links to sequence records, if present, and make sure that each number retrieves the full record of the data.If a new accession number is not linked or a link is broken, provide production staff with the correct URL for the record.If the accession numbers for new data are not publicly accessible before the expected online posting of the article, publication of your article may be delayed; please contact the ASM production staff immediately with the expected release date.
Corresponding authors may join or renew ASM membership to obtain discounts on publication fees.Need to upgrade your membership level?Please contact Customer Service at Service@asmusa.org.
Thank you for submitting your paper to Spectrum.
Sincerely, Ilana Kolodkin-Gal Editor, Microbiology Spectrum Journals Department American Society for Microbiology 1752 N St., NW Washington, DC 20036 E-mail: spectrum@asmusa.org -23R1 (Mechanical Constraints to Unbound Expansion of B. subtilis on Semi-Solid Surfaces) Dear Prof. David Stopar: • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred -23R2 (Mechanical Constraints to Unbound Expansion of B. subtilis on Semi-Solid Surfaces) Dear Prof. David Stopar: • Manuscript: A .DOC version of the revised manuscript • Figures: Editable, high-resolution, individual figure files are required at revision, TIFF or EPS files are preferred